Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a varie ty of agonist-occupied G protein-coupled receptors (GPCR) and plays an impo rtant role in GPCR modulation, However, recent studies suggest additional c ellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind G beta gamma subunits and act as regulators o f G-protein signaling. In this report, we identify phosducin and PhLP as no vel GRK2 substrates, The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P-i/mol of protein, respectively). The phosphorylation reactions exhibit apparent K-m values in the range of 40-100 nM, strongly suggesting that both proteins could be endogenous targets for GRK2 activity . Our data show that the site of phosducin phosphorylation by GRK2 is diffe rent and independent from that previously reported for the cAMP-dependent p rotein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mu tants of phosducin and PhLP indicates that the critical region for GRK2 pho sphorylation is localized in the C-terminal domain of both phosducin and Ph LP (between residues 204 and 245 and 195 and 218, respectively). This regio n is important for the interaction of these proteins with G beta gamma subu nits, Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamm a binding ability, suggesting that GRK2 may modulate the activity of the ph osducin protein family by disrupting this interaction. The identification o f phosducin and PhLP as new substrates for GRK2 further expands the cellula r roles of this kinase and suggests new mechanisms for modulating GPCR sign al transduction.